Medical and Molecular Genetics

Medical and Molecular Genetics

Lecture 10 DNA Synthesis

1)Define the terms primer and template.

Primer – the enzyme that synthesizes DNA(DNA polymerase) can only add nucleotides to an already existing strand of DNA or RNA that is based-paired with the template, i.e. the primer.

Template – The DNA strand that is copied into a complementary strand of DNA.

2)Explain the differences and the similarities between leading and lagging strand synthesis.

Leading Strand / Lagging Strand
Synthesis is 5’ to 3’ / Synthesis is 5’ to 3’
Continuously synthesized / Discontinuously synthesized
Requires an RNA primer / Requires an RNA primer
Primer extention – occurs the same as the lagging strand but only once per template. / Primer extension – DNA polymerase links a dNTP onto the 3’ end of the primer and initiates lagging strand synthesis, its extended for 1,000 nucleotides until it contacts the 5’ end resulting in Okazaki fragments.
Primer removal – also occurs only once to remove the initial primer / Primer removal – the RNaseH degrades the RNA portion of an RNA/DNA duplex and leaves a phosphate group a the 5’ end of the adjoining DNA segment.
DNA polymerase / Gap filling – DNA polymerase uses Okazaki fragment
DNA ligase at the starting primer. / Covalent joining of DNA fragments – the 3’ is joined to the 5’ end by DNA ligase.

3)List the functions of the cis and trans-acting factors required for DNA replication.Multiple origins of replication in eukaryotes are cis-acting factors that create bidirectional replication forks. Trans-acting factors include: Origin binding protein regognizes the origin, denatures the DNA and binds to helicase; Helicase unwinds the DNA double strand; Single-strand DNA binding protein (SSB) prevents the unwound DNA from renaturing; Primase synthesizes RNA primers; DNA polymerase replicates the template; Processivity factors help load and anchor the polymerase; Topoisomerase removes the positive supercoils that form as the fork is unwound; RNaseH removes RNA portions from Okasaki fragments; Ligase seals the nicks.

4)Describe how leading and lagging strand synthesis is coordinated.The two polymerases pointing in opposite directions are held together by a processivity platform and each is bound to a second processivity factor. When the lagging strand finishes an Okasaki fragment it disassociates and is directed to bind to the next primer by another processivity factor.

5)Define what is meant by semidiscontinuous and semiconservative replication. Semiconservative replication means that each of the two new duplexes consist of one of the original parental strands (conserved) and a newly synthesized daughter strand. Semicontinuous replication means the leading strand is continuously replicated while the lagging strand is discontinuously replicated.

6)State the function of telomerase and describe its probable mechanism.Telomerase replicates chromosomal tips by extending the 3’ ends of a chromosome by adding numerous repeats of a six base pair sequence, which is primed by primase and extended by polymerase. The telomerase recognizes the tips of chromosomes with the sequence [TTAGGG]n. Telomerase consists of an RNA sequence complimentary to TTAGGG and a protein. The telomerase binds to a two base portion of the sequence on the 3’ end of the DNA and adds TTAGGG before translocating 6 bases and adding another sequence.

7)List some examples of drugs that inhibit replication and state their mechanism of action.Substrate analogs (dNTPS) can be incorporated into DNA by polymerase and end replication due to their 3’ hydroxyl group which cannot bind:Azidothymidine (AZT) terminates replication by replacing the 3’ hydroxyl group. Acyclovir in an effective anti-herpes virus drug because the herpes thymidine kinase phosphorylates the drug and lets it incorporate into its DNA thus stopping replication. Intercalating agents are generally aromatic compounds that wedge themselves into DNA and prevent the replication complex from binding thus producing mutagenic errors: Anthracycline glycosides and actinomycin D.DNA damaging agents like Cisplatin, which creates a platinum complex that cross-links adjacent guanine residues, causes chemical damage. Topoisomerase inhibitors: Nalidixic acid and fluoroquinolones (bacterial), and campothectin (anti-cancer).